CN117757710A - A high-density fermentation of low-endotoxin Escherichia coli and its application - Google Patents

Abstract

According to the high-density fermentation low endotoxin escherichia coli and the application thereof, lpxP, lpxL, lpxM, pagP genes for regulating and controlling fatty acid chain synthesis on escherichia coli TAK genome, gutQ genes for participating in Kdo molecule synthesis and eptA genes for participating in phosphate group modification are knocked out, so that cells generate Lipid IV without endotoxin activity _A A molecule. In order to improve the growth state of engineering strains under the condition of Kdo synthetic gene deletion, the invention carries out point mutation on msbA genes encoding lipopolysaccharide transporter. The CleanTAK series engineering strain endotoxin prepared by the invention is obviously reduced, and the engineering strain can be used for expressing recombinant protein medicines, vaccine antigens, antibodies, food proteins, enzymes, spider silk and other foreign proteins in the follow-up process, or is used as a host for cloning and producing plasmids, or is used as a chassis for constructing engineering bacteria for synthesizing secondary metabolites, so that the purification steps of proteins, nucleic acids and secondary products are simplified, and the safety of products is improved.

Description

A high-density fermentation of low-endotoxin Escherichia coli and its application

This application is a divisional application of a patent application with a filing date of March 16, 2023, an application number of 202310253436.0, and an invention titled “A low-endotoxin E. coli for high-density fermentation and its application”.

Technical field

The invention relates to the field of bioengineering technology, and specifically to a high-density fermentation of low-endotoxin Escherichia coli and its application.

Background technique

Endotoxins, also known as lipopolysaccharides, are a component of the cell wall of Gram-negative bacteria and are released into the surrounding environment as bacteria grow, divide, die or lyse. The chemical structure of endotoxin is mainly composed of short-chain non-repeating core polysaccharides, long-chain O-specific antigen polysaccharides and hydrophobic lipid A. Lipid A is the active center of endotoxin, including two phosphate groups and six fatty acid chains. This structure can most effectively stimulate the body to produce an immune inflammatory response. At the same time, the length, number and position of the fatty acid chains combined on the carbon skeleton The 1' and 4' phosphate groups are important factors that affect lipid A's stimulation of the TLR4/MD2 pathway and the caspase-11 pathway. Lipid A activates the body's TLR4/MD2/CD14 pathway, stimulating the body to secrete a variety of cytokines, leading to an immune response. In severe cases, it can lead to septic shock or even death (Nat Rev Microbio 2010, 8, 8-14).

In Escherichia coli, multiple enzymes participate in catalyzing the lipopolysaccharide synthesis pathway. Lipid IV _A molecule is a non-glycosylated precursor substance in the synthesis process of lipopolysaccharide. After adding two molecules of Kdo molecules catalyzed by isomerase KdsD or GutQ to its skeleton, the Kdo2-Lipid IV _A molecule is obtained (JBacteriol 2005, 187, 6936–42). On this basis, lauroyltransferase LpxL (HtrB) and myristyltransferase LpxM (MsbB) introduce a fatty acid chain containing 12 carbons and 14 carbons respectively to form the Kdo2-LipidA molecule. Under low temperature (12°C) conditions, Kdo2-LipidIV _A palmitoleoyl-ACP acyltransferase LpxP is induced to express, adding an unsaturated hexacarbonyl chain at the expense of laurate (JBiol Chem 2002, 277, 14186– 93). There are various modifications during lipopolysaccharide synthesis. For example, palmitoyltransferase PagP can add a hexacarbon fatty acid chain to LipidA (lipid A); ethanolamine phosphotransferase EptA can add an ethanolamine phosphate group to LipidA ( Annu Rev Biochem 2007, 76, 295–329). The ABC transporter MsbA is involved in the transport of the core oligosaccharide-LipidA molecule during the synthesis of lipopolysaccharide. The mutation of base 52 or 148 of msbA was confirmed to be an inhibitor of the Kdo deletion mutant (MolMicrobiol 2008, 67, 633–48).

The E. coli TAK strain is a prokaryotic expression strain capable of high-density fermentation and is used for large-scale production of recombinant proteins. However, endotoxin residues have limited the application of recombinant protein products in the pharmaceutical field, and the current endotoxin removal technology in recombinant protein products generally has the disadvantages of high cost, cumbersome operation, and incomplete removal. Therefore, the present invention chooses to use gene editing technology to modify the lipopolysaccharide synthesis or modification pathway to minimize the endotoxin activity of the E. coli expression system itself from the source.

Contents of the invention

The purpose of the present invention is to provide a high-density fermentation of low endotoxin Escherichia coli and its application to solve the problems raised in the above background technology. Specifically, gene knockout technology is used so that the E. coli strain does not contain genes related to fatty acid chain transferase synthesis, and does not contain related genes involved in the synthesis of Kdo molecules and modification of phosphate groups: lpxP, lpxL, lpxM, pagP, kdsD , gutQ, eptA; at the same time, the coding gene of lipopolysaccharide transporter MsbA is carried out point mutation to obtain the low endotoxin Escherichia coli prokaryotic expression system CleanTAK series engineering strains of the present invention. Furthermore, the relevant engineering strains were named CleanTAK 3α, CleanTAK4α, CleanTAK 5α, CleanTAK6α, CleanTAK 7, and CleanTAK 7m. Among them, CleanTAK 3α is an engineering strain that knocks out eptA, kdsD, and pagP genes; CleanTAK 4α is an engineering strain that knocks out eptA, kdsD, pagP, and lpxL genes; CleanTAK5α is an engineering strain that knocks out eptA, kdsD, pagP, lpxL, and lpxP genes. strain; CleanTAK 6α is an engineering strain that knocks out eptA, kdsD, pagP, lpxL, lpxP and lpxM genes; CleanTAK 7 is an engineering strain that knocks out eptA, kdsD, pagP, lpxL, lpxP, lpxM and gutQ genes; CleanTAK 7m is an engineering strain that knocks out eptA, kdsD, pagP, lpxM and gutQ genes. Engineering strains with point mutations in eptA, kdsD, pagP, lpxL, lpxP, lpxM and gutQ genes and msbA gene.

In order to achieve the above objects, the present invention proposes the following technical solutions:

One of the objects of the present invention is to provide a high-density fermented low-endotoxin Escherichia coli and its application. The low-endotoxin Escherichia coli is classified as Escherichia coli CleanTAK 5α and is deposited in the China Typical Culture Collection Center with the preservation number: CCTCC M 20221904, deposited on December 9, 2022.

The invention also provides the application of Escherichia coli CleanTAK 5α in expressing protein/polypeptide substances, including but not limited to protein/polypeptide drugs, protein/polypeptide reagents, protein/polypeptide foods (such as sweet proteins, artificial meat, etc.) , as well as protein/peptide biomaterials such as spider silk.

The present invention also provides the application of Escherichia coli CleanTAK 5α for preparing enzyme products, including but not limited to protease products (such as carboxypeptidase, trypsin, enterokinase, etc.), tool enzymes for in vitro synthesis of mRNA (such as T7 RNA polymerase, Methyltransferase, vaccinia virus capping enzyme, inorganic pyrophosphatase, DNase I, RNase inhibitor, universal nuclease, etc.), feed enzymes (protease, phytase, cellulase, hemicellulase, etc.), biological Chemical enzymes (methylase, racemase, acylase, etc.), etc.

The invention also provides the application of Escherichia coli CleanTAK 5α in preparing plasmids, such as for gene therapy, DNA vaccine, preparation of mRNA, etc.

The invention also provides the Escherichia coli CleanTAK 5α synthetic biology chassis for preparing secondary metabolites, such as polyhydroxyalkanoate, artemisinic acid, taxadiene, n-butanol, etc.

The second object of the present invention is to provide a high-density fermented low-endotoxin Escherichia coli and its application. The low-endotoxin Escherichia coli is classified as Escherichia coli CleanTAK 6α and is deposited in the China Typical Culture Collection Center with the preservation number: CCTCC M 20221905, deposited on December 9, 2022.

The invention also provides the application of Escherichia coli CleanTAK 6α in expressing protein/polypeptide substances, including but not limited to protein/polypeptide drugs, protein/polypeptide reagents, protein/polypeptide foods (such as sweet proteins, artificial meat, etc.) , and protein/peptide biomaterials such as spider silk.

The present invention also provides the application of Escherichia coli CleanTAK 6α for preparing enzyme products, including but not limited to protease products (such as carboxypeptidase, trypsin, enterokinase, etc.), tool enzymes for in vitro synthesis of mRNA (such as T7 RNA polymerase, Methyltransferase, vaccinia virus capping enzyme, inorganic pyrophosphatase, DNase I, RNase inhibitor, universal nuclease, etc.), feed enzymes (protease, phytase, cellulase, hemicellulase, etc.), biological Chemical enzymes (methylase, racemase, acylase, etc.), etc.

The invention also provides the application of Escherichia coli CleanTAK 6α in preparing plasmids, such as for gene therapy, DNA vaccine, preparation of mRNA, etc.

The invention also provides a chassis for Escherichia coli CleanTAK 6α used in synthetic biology to prepare secondary metabolites, such as polyhydroxyalkanoate, artemisinic acid, taxadiene, n-butanol, etc.

The third object of the present invention is to provide a high-density fermentation of low-endotoxin Escherichia coli and its application. The classification name of the low-endotoxin Escherichia coli is Escherichia coli CleanTAK 7m, which is deposited in the China Typical Culture Collection Center with the preservation number: CCTCC M 20221906, deposited on December 9, 2022.

The present invention also provides the application of Escherichia coli CleanTAK 7m for expressing protein/polypeptide substances, including but not limited to protein/polypeptide drugs, protein/polypeptide reagents, protein/polypeptide foods (such as sweet protein, artificial meat, etc.) , as well as protein/peptide biomaterials such as spider silk.

The present invention also provides the application of Escherichia coli CleanTAK 7m for preparing enzyme products, including but not limited to protease products (such as carboxypeptidase, trypsin, enterokinase, etc.), tool enzymes for in vitro synthesis of mRNA (such as T7 RNA polymerase, Methyltransferase, vaccinia virus capping enzyme, inorganic pyrophosphatase, DNase I, RNase inhibitor, universal nuclease, etc.), feed enzymes (protease, phytase, cellulase, hemicellulase, etc.), biological Chemical enzymes (methylase, racemase, acylase, etc.), etc.

The present invention also provides the application of Escherichia coli CleanTAK 7m in preparing plasmids, such as for gene therapy, DNA vaccine, preparation of mRNA, etc.

The invention also provides a chassis for Escherichia coli CleanTAK 7m used in synthetic biology for preparing secondary metabolites, such as polyhydroxyalkanoate, artemisinic acid, taxadiene, n-butanol, etc.

Compared with the existing technology, the main advantages of the present invention are:

1. The E. coli TAK strain is an expression host strain that can be fermented at high density. However, due to the influence of endotoxins during application production, the expression of recombinant proteins is restricted, which in turn affects the application of the E. coli TAK strain in the biomedical industry. The present invention uses gene editing methods to modify genes related to lipopolysaccharide synthesis, modification, and lipopolysaccharide transport, thereby reducing the lipopolysaccharide content. The endotoxin content of the obtained engineering strain is significantly reduced, and the recombinant proteins, plasmids, or The cost of removing endotoxins from raw metabolites thereby reduces the cost of producing recombinant proteins, plasmids or secondary metabolite products, while improving the safety of recombinant protein, plasmid or secondary metabolite products.

2. The low endotoxin activity CleanTAK series engineering strains provided by the present invention still retain the high-density fermentation, rapid growth, and efficient expression of various proteins or synthetic plasmids of the parent TAK strain, but at the same time significantly reduce the endotoxin content, which can be used Produce various high-quality proteins/peptides, nucleic acids or secondary metabolites at low cost. Although its growth rate is inhibited to a certain extent with the increase in the number of gene edits, when it is used to express proteins, its ability to express proteins is no different from the parent strain.

It is to be understood that all combinations of the foregoing concepts as well as additional concepts described in more detail below can be considered part of the disclosure of the present subject matter as long as such concepts are not inconsistent with each other.

The foregoing and other aspects, embodiments and features of the present teachings will be more fully understood from the following description. Additional aspects of the invention, such as features and/or advantages of the exemplary embodiments, will be apparent from the description which follows, or may be learned by practice of specific embodiments in accordance with the teachings of the invention.

Description of the drawings

Figure 1: shows the PCR identification and electrophoresis results of the CleanTAK series engineering strains of the low endotoxin Escherichia coli prokaryotic expression system constructed in the present invention;

Figure 2: is the detection result of whole cell endotoxin activity of CleanTAK series engineering strains in the present invention;

Figure 3: shows the growth characteristics results of the CleanTAK series engineering strains during high-density fermentation in the present invention;

Figure 4: is the result of high-resolution mass spectrometry after purification of lipid A of the CleanTAK series of engineering strains in the present invention; Figure 5: is the expression result of proinsulin glargine protein of the CleanTAK series of engineering strains containing pEINGL expression plasmid in the present invention.

Detailed ways

The present invention will be described in detail below through examples. It is necessary to point out here that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of the present invention. Those skilled in the field will rely on the above content of the invention. Some non-essential improvements and adjustments made still belong to the protection scope of the present invention.

The invention provides a high-density fermentation of low-endotoxin Escherichia coli and its application. Specifically, the low-endotoxin Escherichia coli strain does not contain genes related to fatty acid chain transferase synthesis, and does not contain genes involved in the synthesis of Kdo molecules. Genes related to phosphate group modification: lpxP, lpxL, lpxM, pagP, kdsD, gutQ, eptA. At the same time, point mutations were performed on the gene encoding the lipopolysaccharide transporter MsbA to obtain CleanTAK 3α, CleanTAK 4α, CleanTAK 5α, CleanTAK 6α, and CleanTAK 7m engineering strains. Among them, CleanTAK 3α is an engineering strain that knocks out eptA, kdsD, and pagP genes; CleanTAK4α is an engineering strain that knocks out eptA, kdsD, pagP, and lpxL genes; CleanTAK 5α is an engineering strain that knocks out eptA, kdsD, pagP, lpxL, and lpxP genes. strain; CleanTAK 6α is an engineering strain that knocks out eptA, kdsD, pagP, lpxL, lpxP and lpxM genes; CleanTAK 7m is an engineering strain that knocks out eptA, kdsD, pagP, lpxL, lpxP, lpxM and gutQ genes as well as msbA gene point mutation. . The relevant gene nucleotide sequence information table is as follows:

sequence name Nucleotide sequence ptA SEQ ID No.1 ikB SEQ ID No.2 pgP SEQ ID No.3 wxya SEQ ID No.4 PPx SEQ ID No.5 lm w SEQ ID No.6 gutQ SEQ ID No.7 ikB SEQ ID No.8

Further, a method for constructing high-density fermentation of low-endotoxin E. coli includes the following steps:

1) Heat-shock the thermosensitive pCas plasmid into E. coli TAK cells, and screen for kanamycin (50 μg/mL) resistance to obtain positive clones;

2) Construct pTarget plasmid to express targeting sgRNA;

3) Amplify the upstream and downstream homology arms of the target gene and fuse them through SOE PCR;

4) Electroporate the plasmid obtained in step 2) and the homology arm fragment obtained in step 3) into the strain obtained in step 1), and screen for double resistance to spectinomycin (50 μg/mL) and kanamycin to obtain positive clones ;

5) Obtain engineering strains that knock out the target gene through PCR screening;

6) Eliminate pTarget plasmid and pCas plasmid.

In this way, through CRISPR-Cas9 gene editing technology (AEM 2015, 8, 2506-14), the genes involved in regulating the synthesis of lipopolysaccharide and modification-related enzymes on the genome of the E. coli TAK strain were knocked out, and low endotoxin prokaryotic expression of E. coli was obtained System CleanTAK series engineering strains. The CleanTAK series engineering strain of the low endotoxin Escherichia coli prokaryotic expression system lacks the lpxP, lpxL, lpxM, and pagP genes that regulate the synthesis of lipid A fatty acid chains, thereby making the lipid A structure of the engineering strain contain four fatty acids. The structure of the chain; in addition, it does not contain the gutQ gene involved in the synthesis of Kdo molecules and the eptA gene that does not contain phosphate group modifications, so that the engineered strain can only synthesize Lipid IVA molecules without endotoxin activity.

Point mutations in the gene encoding the lipopolysaccharide transporter MsbA in the above-mentioned gene-deleted strain allowed the engineered strain lacking the Kdo molecule to grow normally. At the same time, the synthesized lipopolysaccharide molecule has the characteristics of low toxicity and easy removal.

Example 1

A high-density fermentation of low-endotoxin E. coli. Through CRISPR-Cas9 gene editing technology, the genes involved in regulating the synthesis of lipopolysaccharide synthesis and modification-related enzymes on the genome of the E. coli TAK strain were knocked out to obtain the CleanTAK series of engineering strains, which specifically include Following steps:

1) Heat-shock the thermosensitive pCas plasmid into E. coli TAK cells, and screen for kanamycin (50 μg/mL) resistance to obtain positive clones;

2) Construct pTarget plasmid to express targeting sgRNA;

3) Amplify the upstream and downstream homology arms of the target gene and fuse them through SOE PCR;

4) Electroporate the plasmid obtained in step 2) and the homology arm fragment obtained in step 3) into the strain obtained in step 1), and screen for double resistance to spectinomycin (50 μg/mL) and kanamycin to obtain positive clones ;

5) Obtain engineering strains that knock out the target gene through PCR screening;

6) Eliminate pTarget plasmid and pCas plasmid.

Further, taking the knockout of the eptA gene as an example, the specific steps for constructing the CleanTAK series of engineering strains are as follows:

1. Transformation of pCas plasmid

Prepare chemically competent cells of the E.coliTAK strain, use heat shock transformation method to transform the pCas plasmid, spread it onto an LB plate containing 50 μg/mL kanamycin, culture it overnight in a 30°C incubator, and pick it from the plate. Streak a single colony onto a new kanamycin-resistant plate. The amplification primers were designed using Snap Gene software and synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The primer sequences are as follows:

S-pCas-F:5’TATCGGCACAAATAGCGTCGGGATGG 3’

S-pCas-R:5’GCGCTAAGGCCAAATAGATTAAGCG 3’

The genomic DNA of the above-mentioned streaked single colony was extracted as a template for PCR amplification verification. The PCR amplification reaction was performed in a 20 μL system. The reaction system was as follows: 2 μL of template DNA, 2× high-fidelity DNase premix, purchased from Novozymes (Nanjing) Biotechnology Co., Ltd. 10 μL, upstream primer 1 μL, downstream primer 1 μL, ddH ₂ O 6 μL. The amplification conditions are: pre-denature at 94°C for 5 minutes before entering the cycle. The cycle parameters are 94°C for 30sec, 53°C for 30sec, and 72°C for 30sec. After 30 cycles, extend at 72°C for 10 minutes. The amplified PCR product was analyzed by 0.8% agarose gel electrophoresis, and the size of the amplified fragment was 450 bp, which was equivalent to the expected size. The selected positive transformants were named TAK(pCas) and stored in a -80°C refrigerator.

2. Construction of pTarget-sgRNA-eptA recombinant plasmid

Search for the 5'-NGG sequence in the 5'UTR region of the eptA gene sequence. Use the 20 bp upstream of the NGG sequence as the targeting sequence of sgRNA. Use Snap Gene software to design the following primers that contain 20 bp and have a 20-25 bp homologous fragment with the pTarget plasmid. , genetically synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.:

eptA-sg-IF:

5’CTCTTTGAATTTACTCGCCGTTTTAGAGCTAGAAATAGCAAGTT 3’

eptA-sg-IR: 5’TCGATGACGCCAACTACCTCTGATA 3’

eptA-sg-VF: 5’TCAGAGGTAGTTGGCGTCATCGAGC 3’

eptA-sg-VR:

5’CAGGCGAGTAAATTCAAAGAACTAGTATTATACCTAGGACTGAGC 3’

Using pTarget plasmid DNA as a template, PCR amplification was performed to construct the insert fragment (Insert) and vector (Vector) fragment of the recombinant plasmid. The PCR amplification reaction was carried out in a 20 μL system. The reaction system was as follows: 2 μL of template DNA, 10 μL of 2× high-fidelity DNase master mix purchased from Novozym (Nanjing) Biotechnology Co., Ltd., 1 μL of upstream primer, and 1 μL of downstream primer. ddH ₂ O 6 μL. Amplification conditions are: pre-denature at 94°C for 5 minutes before entering the cycle. The cycle parameters are 94°C for 30 seconds, 53°C for Insert20sec and Vector2 min, and 72°C for 30 seconds. After 30 cycles, extend at 72°C for 10 minutes. The amplified PCR products were analyzed by 0.8% agarose gel electrophoresis, and the sizes of the amplified fragments were 290bp and 1770bp respectively, which were comparable to the expected sizes.

After purifying and recovering the PCR, the obtained Insert and Vector fragments were connected using a one-step cloning experimental kit purchased from Shanghai Yisheng Company. Generally, a 10 μL reaction system, 5 μL of one-step cloning recombinant enzyme master mix, 3 μL of Insert, and 2 μL of Vector were used. Place the reaction solution in a water bath at 50°C for 25 minutes and then immediately place it on ice for 5 minutes. Then heat-shock it and transform it into E.coli DH5α competent cells. Spread it into an LB plate containing 50 μg/mL spectinomycin. Incubate at 37 °C incubator overnight. The next day, single colonies were picked from the plate and streaked onto another fresh spectinomycin-resistant plate. The amplification primers were designed using SnapGene software and synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The primer sequences are as follows:

S-sgRNA-F: 5’CGACCGAGCGCAGCGAGTCA 3’

S-sgRNA-R: 5’CATCTCGAACCGACGTTGCT 3’

The genomic DNA of the above-mentioned streaked single colony was extracted as a template for PCR amplification verification. The PCR amplification reaction was performed in a 20 μL system. The reaction system was as follows: 2 μL of template DNA, 2× high-fidelity DNase premix, purchased from Novozymes (Nanjing) Biotechnology Co., Ltd. 10 μL, upstream primer 1 μL, downstream primer 1 μL, ddH ₂ O 6 μL. The amplification conditions are: pre-denature at 94°C for 5 minutes before entering the cycle. The cycle parameters are 94°C for 30sec, 53°C for 30sec, and 72°C for 30sec. After 30 cycles, extend at 72°C for 10 minutes. The amplified PCR product was analyzed by 0.8% agarose gel electrophoresis, and the size of the amplified fragment was 460 bp, which was equivalent to the expected size. The PCR product was sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing analysis to ensure that the designed 20 bp targeting sequence was constructed in front of the gRNA scaffold of the pTarget plasmid, and the recombinant plasmid pTarget-sgRNA-eptA was successfully constructed. Save the screened positive transformants.

3. Amplification and fusion of the upstream and downstream homology arms of the eptA gene

Two pairs of fusion PCR primers were designed in the TAK genome sequence to amplify the upper and lower homology arms of the eptA gene respectively. The amplified fragment sizes are both 400 bp. The primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The primer sequences are as follows :

eptA-up-F:5’AAAGAGTTCGGTCTGGTTTCTTCCG 3’

eptA-up-R:5’ATCAGAATTTCACGGTGTTTCCATCGAACAAAGTG 3’

eptA-down-F:5’AAACACCGTGAAATTCTGATTGTTGAAGACGATAC 3’

eptA-down-R:5’TTCACGTCAGATTGCCAACAATCA 3’

The genomic DNA of the TAK strain in the logarithmic growth phase was extracted as a template for PCR amplification verification. The PCR amplification reaction was performed in a 20 μL system. The reaction system was as follows: 2 μL of template DNA, 2× high-fidelity DNase premix, purchased from Novo Weizan (Nanjing) Biotechnology Co., Ltd. 10 μL, upstream primer 1 μL, downstream primer 1 μL, ddH ₂ O 6 μL. The amplification conditions are: pre-denature at 94°C for 5 minutes before entering the cycle. The cycle parameters are 94°C for 30sec, 53°C for 30sec, and 72°C for 30sec. After 30 cycles, extend at 72°C for 10 minutes. The amplified PCR product was analyzed by 0.8% agarose gel electrophoresis, and the size of the amplified fragment was 400 bp, which was equivalent to the expected size.

Fusion of upstream and downstream homology arms: Add 1 μL of each of the amplified upstream and downstream homology arms as a template for overlap extension PCR, and use the primers eptA-up-F and eptA-down-R in the same amplification system as before Amplification was carried out under the conditions of pre-denaturation at 94°C for 5 min before entering the cycle. The cycle parameters were 94°C for 30 sec, 53°C for 30 sec, and 72°C for 1 min. After 30 cycles, extend at 72°C for 10 minutes. A homology arm fusion fragment of 800 bp in size was obtained, and the PCR product was purified and recovered using a PCR product purification kit purchased from Thermo Fisher Scientific (China) Co., Ltd.

4. Construction and identification of engineering strains lacking eptA gene

The constructed pTarget-sgRNA-eptA recombinant plasmid and the fused homology arm fragment were electroporated into the E. coli TAK (pCas) strain, spread onto a plate containing spectinomycin and kanamycin double resistance, and placed for 30 °C incubator overnight. Pick a single colony and streak it on a new double-antibody plate for culture, and then extract its genome as a template for PCR amplification verification. The PCR amplification reaction is carried out in a 20 μL system. The reaction system is as follows: template DNA 2 μL, 2× high-security True DNase master mix, purchased from Novozymes (Nanjing) Biotechnology Co., Ltd., 10 μL, upstream primer 1 μL, downstream primer 1 μL, ddH ₂ O 6 μL. The amplification conditions are: pre-denature at 94°C for 5 minutes before entering the cycle. The cycle parameters are 94°C for 30sec, 53°C for 30sec, and 72°C for 30sec. After 30 cycles, extend at 72°C for 10 minutes. The amplified PCR product was analyzed by 0.8% agarose gel electrophoresis, and the size of the amplified fragment was 1000 bp, which was equivalent to the expected size. The primer sequences used are as follows:

U-eptA-F:5’CTGCGGGTTGCTTAGGTTCACTGGG 3’

U-eptA-R:5’TGTTGGTCGAGGGTTCATTGTCCCA 3’

The PCR product with the expected band size was sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing analysis. The sequencing results were compared with the genome of the TAK strain, confirming that the eptA gene was successfully knocked out, and the engineering strain was named CleanTAK eptA. .

5. Elimination of plasmids in CleanTAK eptA engineering strains

The CleanTAK eptA engineering strain containing pTarget and pCas dual plasmids was transferred into 5 mL of LB liquid culture medium containing Kan (50 μg/mL) resistance, and then IPTG with a final concentration of 1 mmol/L was added to induce the sgRNA on the pCas plasmid. Expression, culture overnight in a shaker at 30°C and 180r/min. Since the sgRNA-pBR322 ori binds to the Cas9 protein and cuts the pTarget replication origin, the pTarget plasmid cannot replicate normally, so the pTarget plasmid is lost. Spread the strains cultured overnight onto LB solid plates containing 50 μg/mL kanamycin. After culture, select single colonies and spot them to contain only kan resistance and verify pTarget on plates containing both Spec and Kan dual resistance. Whether the plasmid was successfully eliminated. Select a single colony containing only pCas plasmid and inoculate it into 5 mL of LB medium, and culture it overnight in a shaker at 37°C and 180 r/min. Since the pCas plasmid is a temperature-sensitive plasmid and cannot replicate normally at 37°C, the pCas plasmid was lost and the CleanTAK eptA engineering strain without plasmid was obtained. The strain whose pCas plasmid was not eliminated was stored at -80°C and named CleanTAK eptA (pCas), which was used to construct a double-gene knockout engineering strain.

According to the above method, the constructed recombinant pTarget plasmid of each target gene and the fused homology arm fragment are electroporated into the cells containing the pCas plasmid of the previous gene-deletion engineering strain. CleanTAK 2α, CleanTAK3α, CleanTAK4α, and CleanTAK 5α engineering strains were constructed in sequence. The gene combinations knocked out by each strain are as follows:

CleanTAK 2α engineering strain: ΔeptAΔkdsD

CleanTAK 3α engineering strain: ΔeptAΔkdsDΔpagP

CleanTAK4α engineering strain: ΔeptAΔkdsDΔpagPΔlpxL

CleanTAK 5α engineering strain: ΔeptAΔkdsDΔpagPΔlpxLΔlpxP

Furthermore, the relevant identification PCR results of the engineering strains in this example are shown in Figures 1A-E.

Example 2

Different from the above Example 1, on the basis of the CleanTAK 5α engineering strain, the constructed recombinant pTarget plasmid and the fused homology arm fragment of each target gene were electroporated into the cells of the CleanTAK 5α engineering strain containing the pCas plasmid to construct CleanTAK 6α engineering strain, its knockout gene combination is: ΔeptAΔkdsDΔpagPΔlpxLΔlpxPΔlpxM.

Furthermore, the identification PCR results of the engineering strain in this example are shown in Figures 1A-F.

Example 3

Different from the above Example 1, on the basis of the CleanTAK 6α engineering strain, the constructed recombinant pTarget plasmid and the fused homology arm fragment of each target gene were electroporated into the cells of the CleanTAK 6α engineering strain containing the pCas plasmid to construct CleanTAK 7 engineering strain, its knockout gene combination is: ΔeptAΔkdsDΔpagPΔlpxLΔlpxPΔlpxMΔgutQ.

Furthermore, the engineering strain construction in this example includes the following steps:

1. Construction of pTarget-sgRNA-msbA recombinant plasmid

Search for the 5'-NGG sequence near the site to be mutated in the msbA gene. Use the 31 bp upstream of the NGG sequence as the target sequence of sgRNA. Use SnapGene software to design the following primers containing 20 bp and 20-25 bp homologous fragments with the pTarget plasmid. Gene synthesis of Sangon Bioengineering (Shanghai) Co., Ltd.:

msbA-sg-IF:

5’GATCTGTTTTACCAAAGCCATCATCAAGAAGGTTTTAGAGCTAGAAATA GCAAGTT 3’

msbA-sg-IR: 5’CGATCAACGGCACTGTTGCAAATAG 3’

msbA-sg-VF: 5’CTATTTGCAACAGTGCCGTTGATCG 3’

msbA-sg-VR:

5’TTCTTGATGATGGCTTTGGTAAAACAGATCACTAGTATTATACCTAGGAC TGAGC 3’

Using pTarget plasmid DNA as a template, PCR amplification was performed to construct the insert fragment (Insert) and vector (Vector) fragment of the recombinant plasmid. The specific plasmid construction and verification experimental operations are the same as the construction of pTarget-sgRNA-eptA recombinant plasmid.

2. Amplification and fusion of msbA gene fragments containing point mutation sites

Two pairs of fusion PCR primers were designed to amplify the exogenous fragments required for point mutations in the msbA gene. The primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., and the primer sequences are as follows:

msbA-S1-F:5’AAAAGTGGCACTCGCATCGG 3’

msbA-S1-R:

5’CGATCTGTTTTACCAAAGCCATCATCAAGAAGTGACTTAAG

GAGCGATAACATGAAGGT 3’

msbA-S2-F:

5’ACCTTCATGTTATCGCTCCTTAAGTCACTTCTTGATGATGG

CTTTGGTAAAACAGATCG 3’

msbA-S2-R:5’AAAACGCTTCGATACAACGC 3’

The msbA-S1-R and msbA-S2-F primers contain mutated bases, that is, the base at position 35 is replaced by G/C to A/T.

The genomic DNA of the TAK strain in the logarithmic growth phase was extracted as a template for PCR amplification verification. The PCR amplification reaction was performed in a 20 μL system. The reaction system was as follows: 2 μL of template DNA, 2× high-fidelity DNase premix, purchased from Novo Weizan (Nanjing) Biotechnology Co., Ltd. 10 μL, upstream primer 1 μL, downstream primer 1 μL, ddH ₂ O 6 μL. The amplification conditions are: pre-denature at 94°C for 5 minutes before entering the cycle. The cycle parameters are 94°C for 30sec, 53°C for 30sec, and 72°C for 30sec. After 30 cycles, extend at 72°C for 10 minutes. The amplified PCR product was analyzed by 0.8% agarose gel electrophoresis, and the size of the amplified fragment was 400 bp, which was equivalent to the expected size.

Fusion of S1 and S2 fragments: Add 1 μL of each of the amplified S1 and S2 fragments as a template for overlap extension PCR, and use primers msbA-S1-F and msbA-S2-R to amplify under the same amplification system as before. The amplification conditions were pre-denaturation at 94°C for 5 min before entering the cycle. The cycle parameters were 94°C for 30 sec, 53°C for 30 sec, and 72°C for 1 min. After 30 cycles, extend at 72°C for 10 minutes. A fusion fragment of 800 bp in size was obtained. The PCR product was sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing analysis to ensure that the obtained fragment contained mutated bases. The remaining PCR products were purchased from Thermo Fisher Scientific ( China) Co., Ltd.'s PCR product purification kit for purification and recovery.

3. Construction and identification of msbA gene point mutation engineering strains

The constructed pTarget-sgRNA-msbA recombinant plasmid and the fused exogenous fragment were electrotransduced into the E. coli CleanTAK 7 (pCas) strain, spread on a plate containing spectinomycin and kanamycin double resistance, and placed for 30 °C incubator overnight. Pick a single colony and streak it on a new double-antibody plate for culture, and then extract its genome as a template for PCR amplification verification. The PCR amplification reaction is carried out in a 20 μL system. The reaction system is as follows: template DNA 2 μL, 2× high-security True DNase master mix, purchased from Novozymes (Nanjing) Biotechnology Co., Ltd., 10 μL, upstream primer 1 μL, downstream primer 1 μL, ddH ₂ O 6 μL. The amplification conditions are: pre-denature at 94°C for 5 minutes before entering the cycle. The cycle parameters are 94°C for 30sec, 53°C for 30sec, and 72°C for 30sec. After 30 cycles, extend at 72°C for 10 minutes. The amplified PCR product was analyzed by 0.8% agarose gel electrophoresis, and the size of the amplified fragment was 1000 bp, which was equivalent to the expected size. The primer sequences used are as follows:

U-msbA-F:5’TAACGGGTAGAATATGCGGC 3’

U-msbA-R:5’CTGGCATTCCCATCATGTGA 3’

The PCR product with the expected band size was sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing analysis. The sequencing results were compared with the genome of the TAK strain. The comparison results are shown in Figure 1A-H, confirming the successful point mutation of the msbA gene. The engineered strain was named CleanTAK 7m.

5. Elimination of plasmids in CleanTAK 7m engineering strain

The CleanTAK 7m engineering strain containing the pTarget and pCas dual plasmids was transferred into 5 mL of LB liquid culture medium containing Kan (50 μg/mL) resistance, and then IPTG with a final concentration of 1 mmol/L was added to induce the sgRNA on the pCas plasmid. Expression, culture overnight in a shaker at 30°C and 180r/min. Since the sgRNA-pBR322 ori binds to the Cas9 protein and cuts the pTarget replication origin, the pTarget plasmid cannot replicate normally, so the pTarget plasmid is lost. The strains cultured overnight were spread on LB solid plates with 50 μg/mL kanamycin. After culture, single colonies were selected and inoculated onto plates containing only Kan resistance, and plates containing both Spec and Kan dual resistance. Verify that the pTarget plasmid was successfully eliminated. Select a single colony containing only pCas plasmid and inoculate it into 5 mL of LB medium, and culture it overnight in a shaker at 37°C and 180 r/min. Since the pCas plasmid is a temperature-sensitive plasmid and cannot replicate normally at 37°C, the pCas plasmid was lost and the CleanTAK7m engineering strain without plasmid was obtained.

Example 4

This example relates to the detection of endotoxin activity in whole cells of Escherichia coli CleanTAK series engineering strains. details as follows:

The endotoxin activity detection method of E. coli TAK strains and CleanTAK series engineering strains is as follows: Inoculate each strain to be tested into 5 mL LB liquid culture medium, culture it overnight with shaking at 37°C, and then inoculate 300 μL into fresh 30 mL LB liquid culture medium. Medium, culture at 37°C until OD ₆₀₀ ≈0.7. Take 4 mL of bacterial liquid of each strain, centrifuge at 5000 rpm for 5 min, discard the supernatant, resuspend the precipitate in 4 ml of Watson's distilled water, and centrifuge again at 5000 rpm for 5 min. Repeat three times. After resuspending the pellet in 4 mL of Watson's distilled water, take 2 mL and measure the OD ₆₀₀ value of the bacterial suspension using a spectrophotometer. Based on the OD ₆₀₀ value of 0.52, dilute and adjust each bacterial suspension. Take 1 mL of the bacterial suspension of each strain mentioned above and conduct ultrasonic disruption with a power of 90W and ultrasonic disruption for 10 minutes. The ultrasonically broken solution was centrifuged at 8000 rpm for 30 min. Collect the supernatant. The cell lysate supernatant of each strain was serially diluted, and the bacterial endotoxin detection kit (end-point chromogenic matrix method) purchased from Fuzhou Xinbei Biochemical Industry Co., Ltd. was used to detect the whole-cell endotoxin activity of each strain. For specific experimental methods, see Instructions, I won’t go into details here. Take 200 μL of the endotoxin detection mixture on a microplate, measure the absorbance using a microplate reader at a wavelength of 562nm, and then calculate the endotoxin activity based on the standard curve.

The statistics of the whole-cell endotoxin activity detection results of the engineering strains described in Examples 1 to 3 are shown in Figure 2. With the accumulation of the number of gene edits, the endotoxin content of the obtained engineering strains also showed a downward trend. The endotoxin content of CleanTAK 7m strain The content dropped most significantly, 96.39% lower than that of the wild TAK strain.

Example 5

This example involves the analysis of the growth characteristics of E. coli CleanTAK series engineering strains, as follows:

Take 100 μL of frozen bacterial liquid of each strain from the parent TAK strain and engineering strains with significantly reduced endotoxin activity, CleanTAK 4α, CleanTAK 5α, CleanTAK 6α, CleanTAK 7 and CleanTAK 7m, and transfer it to a 5 mL LB test tube culture medium, and incubate at 33°C and 220 rpm. Culture in a shaker for 15-16h. Take 1000 μL of the above bacterial liquid culture solution and transfer it to 100 mL 1/2TB culture medium, and culture it in a shaker at 33°C and 220 rpm for 5-8 hours (the culture time is determined according to the concentration of the bacteria in the shake flask). Set the fermentation tank fermentation volume to 7L, medium formula: lactose 150g/L, yeast 15g/L, SM 6.5g/L. Transfer the cultured bacterial liquid in the shake flask into the fermentation tank, set the fermentation parameters: temperature 33°C, rotation speed 650rpm, ventilation volume 9.5L/min, tank pressure 0.02MPa, and control the fermentation pH to 6.7 for fermentation culture. During the fermentation process, measure OD ₆₀₀ and bacterial content every 3 hours, and draw the fermentation growth curve as shown in Figure 3. CleanTAK4α, CleanTAK 5α, and CleanTAK6α strains can grow at high density in high-density fermenters. The CleanTAK7 strain in Example 3 lacks The key gene for Kdo synthesis caused its growth to be severely inhibited. The growth status of CleanTAK 7m strain obtained after point mutation of msbA gene was improved.

Example 6

1. Purification of LipidA from E. coli CleanTAK series engineering strains

The parent TAK strain and engineering strains CleanTAK 5α, CleanTAK 6α and CleanTAK 7m, which have significantly reduced endotoxin activity and good growth characteristics, were activated and cultured overnight. The next day, they were transferred to fresh 200mL LB medium at a ratio of 1:100, at 37°C. , cultivate at 180r/min until OD ₆₀₀ is between 0.8-0.9. Collect the cells by centrifugation for 10 minutes at 4°C, wash once with phosphate buffer, resuspend the bacterial pellet in 20 mL of phosphoric acid, and add chloroform methanol. The final ratio is chloroform/methanol/water = 1:2:0.8 (v/v) to form Bligh /Dyer single-phase system. Shake the bacterial suspension at room temperature for 1 hour, then centrifuge at 2500×g for 20 minutes, remove the upper layer containing phospholipids, resuspend the pellet in 40mL of single-phase Bligh/Dyer, transfer to a 50mL centrifuge tube, centrifuge, discard the supernatant, and precipitate air After drying, resuspend in 25 mL of 50 mM, pH 4.5 sodium acetate solution (dissolve in the solution by shaking and ultrasonic waves). If necessary, use acetic acid to adjust the pH to 4.5, and then release LipidA in a boiling water bath for 30 minutes. After shaking and cooling, transfer the solution to a centrifuge tube, add chloroform methanol to a volume ratio of chloroform/methanol/water = 2:2:1.8 to form a Bligh/Dyermixture two-phase system, shake vigorously enough to extract LipidA, and then 2500×g, Centrifuge at 20°C for 20 minutes to form two phases. Carefully collect the lower phase (chloroform organic phase) into a round-bottomed flask. Add an equal amount of chloroform to the solution in the centrifuge tube to form a two-phase again. Carefully collect the lower phase after centrifugation. Add to the same round-bottomed flask, remove the chloroform by rotary evaporation, dissolve the sample in chloroform/methanol = 2:1 (v/v), transfer to a small centrifuge tube and dry, cover tightly and store at -80°C.

2. ESI/MS analysis of LipidA of Clean TAK series engineering strains

Mass spectrometry detection of all samples was performed on a WATERS G2-XS Qtof high-resolution mass spectrometer. The purified lipid A was dissolved in chloroform:methanol=2:1 (v/v). Using ESI ion source, positive ion detection mode, the detection range is less than m/z 2500, and nitrogen is used as the collision gas. The final results are shown in Figure 4. The results show that the lipid A structure of the E. coli TAK strain can be successfully transformed by knocking out a series of genes such as eptA and kdsD, and the change in the lipid A structure makes the endotoxin activity of the engineered strain decreased, which was consistent with the detection results in Example 4, laying a theoretical foundation for the subsequent application of low endotoxin activity engineering strains.

Example 7

This example involves analysis of the protein expression ability of CleanTAK series engineering strains, specifically as follows:

The pEINGL expression plasmid was transformed into E. coli TAK, CleanTAK 5α, CleanTAK 6α, and CleanTAK 7m engineering strains, and activated and transferred into 1/2TB medium containing lactose to induce the expression of proinsulin glargine protein. Take an appropriate amount of bacterial solution and concentrate it until the OD ₆₀₀ reading is 20, mix well; take 40 μL of each sample and add 10 μL buffer, mix and boil in boiling water for 5 minutes; centrifuge at 12000 rpm for 5 minutes in a desktop centrifuge to prepare 14% sodium lauryl sulfate- Polyacrylamide gel electrophoresis was observed. According to the SDS-PAGE electrophoresis results, it can be seen that compared with the parent strain TAK, there is no significant difference in the protein expression ability of the three engineering strains: CleanTAK 5α engineering strain, CleanTAK6α engineering strain, and CleanTAK 7m engineering strain. See Figure 5.

sequence list

Sequence Listing

ptA

1

1644

DNA

Escherichia coli str.K-12substr.MG1655

1atgttgaagc gcctactaaa aagaccctct ttgaatttac tcgcctggct attgttggcc

61gctttttata tctctatctg cctgaatatt gcctttttta aacaggtgtt gcaggcgctg

121ccgctggatt cgctgcataa cgtactggtt ttcttgtcga tgccggtcgt cgctttcagc

181gtgattaata ttgtcctgac actaagctct ttcttatggc ttaatcgacc actggcctgc

241ctgtttatattc tggttggcgc ggctgcacaa tatttcataa tgacttacgg catcgtcatc

301gaccgctcga tgattgccaa tattattgat accactccgg cagaaagtta tgcgctgatg

361acaccgcaaa tgttattaac gctgggattc agcggcgtgc ttgctgcgct gattgcctgc

421tggataaaaa tcaaacctgc cacctcgcgt ctgcgcagtg ttcttttccg tggagccaat

481attctggttt ctgtactact gattttgctg gtcgccgcac tgttttataa agactacgcc

541tcgttgttcc gcaataacaa agagctggtg aaatccttaa gcccctctaa cagcattgtt

601gccagctggt catggtactc ccatcagcga ctggcaaatc tgccgctggt gcgaattggt

661gaagacgcgc accgcaaccc gttaatgcag aacgaaaaac gtaaaaattt gaccatcctg

721attgtcggcg aaacctcgcg ggcggagaac ttctccctca acggctaccc gcgtgaaact

781aacccgcggc tggcgaaaga taacgtggtc tatttcccta ataccgcatc ttgcggcacg

841gcaacggcag tttcagtacc gtgcatgttc tcggatatgc cgcgtgagca ctacaaagaa

901 gagctggcac agcaccagga aggcgtgctg gatatcattc agcgagcggg catcaacgtg

961 ctgtggaatg acaacgatgg cggctgtaaa ggtgcctgcg accgcgtgcc tcaccagaac

1021 gtcaccgcgc tgaatctacc tgatcagtgc atcaacggcg aatgctatgacgaagtgctg

1081 ttccacgggc ttgaagagta catcaataac ctgcaaggtg atggcgtgattgtcttacac

1141 accatcggca gccacggtcc gacctattac aaccgctatc cgcctcagttcaggaaattt

1201 accccaacct gcgacaccaa tgagatccag acctgtacca aagagcaactggtgaacact

1261 tacgacaaca cgctggttta cgtcgactat attgttgata aagcgattaatctgctgaaa

1321 gaacatcagg ataaatttac caccagcctg gtttatcttt ctgaccacggtgaatcgtta

1381 ggtgaaaatg gcatctatct gcacggtctg ccttatgcca tcgccccggatagccaaaaa

1441 caggtgccga tgctgctgtg gctgtcggag gattatcaaa aacggtatcaggttgaccag

1501 aactgcctgc aaaaacaggc gcaaacgcaa cactattcac aagacaatttattctccacg

1561 ctattgggat taactggcgt tgagacgaag tattaccagg ctgcggatgatattctgcaa

1621 acttgcagga gagtgagtga atga

ikB

2

987

DNA

Escherichia coli str.K-12 substr.MG1655

1 atgtcgcacg tagagttaca accgggtttt gactttcagc aagcaggtaa agaagtcctg

61 gcgattgaac gtgaatgcct ggcggagctt gatcaataca tcaatcagaa tttcacgctt

121 gcctgtgaaa agatgttctg gtgtaaaggg aaagttgtcg tcatggggat gggaaaatcg

181 gggcatattg ggcgaaaaat ggcggcaacg tttgccagca ccggtacacc ttcatttttc

241 gtccatcctg gtgaagccgc gcatggtgat ttaggcatgg ttaccccaca ggatgtggtg

301 attgctatct ctaactctgg tgaatccagc gaaatcacgg ccttaattcc agtgcttaag

361 cgtcttcacg taccgttaat ctgcatcacc ggtcgcccgg agagcagcat ggcgcgcgcc

421 gcagatgtgc atctgtgtgt taaagtagcg aaagaagcct gtccgttagg gctggcaccg

481 accagcagca ccaccgccac gctggttatg ggcgatgccc tcgctgtcgc gctgttaaaa

541 gcacgcggct ttactgctga agattttgcg ctctcacacc caggcggcgc actgggtcgt

601 aaacttctgc tgcgcgtaaa cgatattatg catacgggcg atgagatccc gcatgttaag

661 aaaacggcca gtctgcgtga cgcgttgctg gaagttaccc gcaaaaatct tggtatgact

721 gtcatttgcg atgacaatat gatgattgaa ggcatcttta ccgacggtga tttacgccgt

781 gtcttcgata tgggcgtgga tgttcgtcag ttaagtattg ccgatgtgat gacgccgggg

841 ggaatacgtg tgcgccctgg cattctggcc gttgaggcac tgaacttaat gcagtcccgc

901 catatcacct ccgtgatggt tgccgatggc gaccatttac tcggtgtgtt acatatgcat

961 gatttactgc gtgcaggcgt agtgtaa

pgP

3

561

DNA

Escherichia coli str.K-12 substr.MG1655

1 atgaacgtga gtaaatatgt cgctatcttt tcctttgttt ttattcagtt aatcagcgtt

61 ggtaaagttt ttgctaacgc agatgagtgg atgacaacgt ttagagaaaa tattgcacaa

121 acctggcaac agcctgaaca ttatgattta tatattcctg ccatcacctg gcatgcacgt

181 ttcgcttacg acaaagaaaa aaccgatcgc tataacgagc gaccgtgggg tggcggtttt

241 ggcctgtcgc gttggggatga aaaaggaaac tggcatggcc tgtatgccat ggcatttaag

301 gactcgtgga acaaatggga accgattgcc ggatacggat gggaaagtac ctggcgaccg

361 ctggcggatg aaaattttca tttaggtctg ggattcaccg ctggcgtaac ggcacgcgat

421 aactggaatt acatccctct cccggttcta ctgccattgg cctccgtggg ttatggccca

481 gtgacttttc agatgaccta cattccgggt acctacaaca atggcaatgt gtactttgcc

541 tggatgcgct ttcagttttg a

wxya

4

921

DNA

Escherichia coli str.K-12 substr.MG1655

1 atgacgaatc tacccaagtt ctccaccgca ctgcttcatc cgcgttatttg gttaacctgg

61 ttgggtattg gcgtactttg gttagtcgtg caattgccct acccggttat ctaccgcctc

121 ggttgtggat taggaaaact ggcgttacgt tttatgaaac gacgcgcaaa aattgtgcat

181 cgcaacctgg aactgtgctt cccggaaatg agcgaacaag aacgccgtaa aatggtggtg

241 aagaatttcg aatccgttgg catgggcctg atggaaaccg gcatggcgtg gttctggccg

301 gaccgccgaa tcgcccgctg gacggaagtg atcggcatgg aacacattcg tgacgtgcag

361 gcgcaaaaac gcggcatcct gttagttggc atccattttc tgacactgga gctgggtgcg

421 cggcagtttg gtatgcagga accgggtatt ggcgtttatc gcccgaacga taatccactg

481 attgactggc tacaaacctg gggccgtttg cgctcaaata aatcgatgct cgaccgcaaa

541 gatttaaaag gcatgattaa agccctgaaa aaaggcgaag tggtctggta cgcaccggat

601 catgattacg gcccgcgctc aagcgttttc gtcccgttgt ttgccgttga gcaggctgcg

661 accacgaccg gaacctggat gctggcacgg atgtccggcg catgtctggt gcccttcgtt

721 ccacgccgta agccagatgg caaagggtat caattgatta tgctgccgcc agagtgttct

781 ccgccactgg atgatgccga aactaccgcc gcgtggatga acaaagtggt cgaaaaatgc

841 atcatgatgg caccagagca gtatatgtgg ttacaccgtc gctttaaaac acgcccggaa

901 ggcgttcctt cacgctatta a

PPx

5

921

DNA

Escherichia coli str.K-12 substr.MG1655

1 atgtttccac aatgcaaatt ttcccgcgag tttctacatc ctcgctactg gctcacatgg

61 tttgggcttg gtgtactctg gctttgggta cagcttcctt atcctgttct ctgctttctc

121 ggcacgcgta ttggcgcaat ggcgcgacca ttcctgaaac gtcgtgaatc tatcgcccgt

181 aaaaacctgg aactttgttt cccgcagcat tctgcggaag aacgcgagaa gatgattgcc

241 gaaaactttc gttcactcgg catggcgctg gtagaaaccg gcatggcatg gttctggccc

301 gacagtcgcg tacgtaaatg gtttgatgtt gaagggttgg ataaccttaa acgcgcacaa

361 atgcaaaatc gcggcgtaat ggttgtcggc gtccatttta tgtcgctgga actgggcggc

421 cgcgtgatgg gactgtgcca accaatgatg gctacctatc gtccacataa taatcagctg

481 atggaatggg tgcagacccg tgggcgcatg cgctctaaca aagcgatgat cggcagaaat

541 aatctgcgcg gcattgtcgg tgcactgaag aaaggtgaag cggtatggtt tgctcccgat

601 caggattatg gtcgtaaagg cagctccttc gcgccgttct ttgcggtgga aaatgtcgcc

661 acaaccaatg gcacctatgt tctctcccgt ctctctggcg cagccatgtt gaccgtaacg

721 atggtaagaa aagcggatta cagcggatat cgtttgttca tcaccccaga gatggaaggc

781 tacccgacag atgaaaatca agccgctgcc tatatgaaca agattatcga gaaagagatc

841 atgcgcgcac cggagcagta cctctggatc caccgtcgct ttaaaacgcg cccggtggga

901 gaatcgtcgt tgtacattta a

lm w

6

972

DNA

Escherichia coli str.K-12 substr.MG1655

1 atggaaacga aaaaaaataa tagcgaatac attcctgagt ttgataaatc ctttcgccac

61 ccgcgctact ggggagcatg gctgggcgta gcagcgatgg cgggtatcgc tttaacgccg

121 ccaaagttcc gtgatcccat tctggcacgg ctgggacgtt ttgccggacg actgggaaaa

181 agctcacgcc gtcgtgcgtt aatcaatctg tcgctctgct ttccagaacg tagtgaagct

241 gaacgcgaag cgattgttga tgagatgttt gccaccgcgc cgcaagcgat ggcaatgatg

301 gctgagttgg caatacgcgg gccggagaaa attcagccgc gcgttgactg gcaagggctg

361 gagatcatcg aagagatgcg gcgtaataac gagaaagtta tctttctggt gccgcacggt

421 tgggccgtcg atattcctgc catgctgatg gcctcgcaag ggcagaaaat ggcagcgatg

481 ttccataatc agggcaaccc ggtttttgat tatgtctgga acacggtgcg tcgtcgcttt

541 ggcggtcgtc tgcatgcgag aaatgacggt attaaaccat tcatccagtc ggtacgtcag

601 gggtactggg gatattattt acccgatcag gatcatggcc cagagcacag cgaatttgtg

661 gatttctttg ccacctataa agcgacgttg cccgcgattg gtcgtttgat gaaagtgtgc

721 cgtgcgcgcg ttgtaccgct gtttccgatt tatgatggca agacgcatcg tctgacgatt

781 caggtgcgcc caccgatgga tgatctgtta gaggcggatg atcatacgat tgcgcggcgg

841 atgaatgaag aagtcgagat ttttgttggt ccgcgaccag aacaatacac ctggatacta

901 aaattgctga aaactcgcaa accgggcgaa atccagccgt ataagcgcaa agatctttat

961cccatcaaataa

gutQ

7

966

DNA

Escherichia coli str.K-12 substr.MG1655

1 atgagtgaag cactactgaa cgcgggacgt cagacgttaa tgctggagtt gcaggaagca

61 agccgtttac cggaacgtct gggcgatgat tttgttcgcg ccgccaatat catcctgcac

121 tgtgaaggca aagtggtggt ttcgggaatt ggcaaatcgg gccacattgg taagaaaatc

181 gccgcaacgc ttgccagtac cggcactccg gctttttttg tccatccggc agaagcgctg

241 cacggcgatc tggggatgat cgaaagccgc gatgtgatgc tgtttatctc ttactccggt

301 ggcgcgaagg aactggatct gattattccg cgtctggaag ataaatctat cgcgctgctg

361 gcgatgaccg gcaaaccgac gtcaccgctg ggcctggcgg caaaagcggt gctggatatc

421 tccgtagaac gcgaagcctg cccgatgcac cttgcgccga cctccagcac cgtcaatacc

481 ctgatgatgg gtgacgcgct ggcgatggcg gtcatgcagg cgcgcggatt taatgaagaa

541 gattttgccc gctcccaccc agccggggca ctgggcgctc gcttgctgaa taaagtgcat

601 catctgatgc gccgtgacga tgccatccca caggtggcgt taaccgccag cgtgatggat

661 gcgatgctgg aactcagccg caccggtctg gggctggtgg cggtatgtga cgctcaacaa

721 caggtacaag gcgtctttac cgacggcgat ttacgtcgct ggctggttgg cggcggcgca

781 ctcaccacgc cagtcaatga agcgatgacg gtcggcggca ccacgttgca atcgcaaagt

841 cgcgccatcg acgccaaaga gatcctgatg aagcgcaaaa tcactgccgc accggtggtg

901 gatgaaaacg gcaaactcac cggcgcaata aacctgcagg atttctatca ggccgggatt

961 atttaa

ikB

8

1749

DNA

Escherichia coli str.K-12 substr.MG1655

1 atgcataacg acaaagatct ctctacgtgg cagacattcc gccgactgtg gccaaccatt

61 gcgcctttca aagcgggtct gatcgtggcg ggcgtagcgt taatcctcaa cgcagccagc

121 gataccttca tgttatcgct ccttaagcca cttcttgatg atggctttgg taaaacagat

181 cgctccgtgc tggtgtggat gccgctggtg gtgatcgggc tgatgatttt acgtggtatc

241 accagctatg tctccagcta ctgtatctcc tgggtatcag gaaaggtggt aatgaccatg

301 cgtcgccgcc tgtttggtca catgatggga atgccagttt cattctttga caaacagtca

361 acgggtacgc tgttgtcacg tattacctac gattccgaac aggttgcttc ttcttcttcc

421 ggcgcactga ttactgttgt gcgtgaaggt gcgtcgatca tcggcctgtt catcatgatg

481 ttctattaca gttggcaact gtcgatcatt ttgattgtgc tggcaccgat tgtttcgatt

541 gcgattcgcg ttgtatcgaa gcgtttttcgc aacatcagta aaaacatgca gaacaccatg

601 gggcaggtga ccaccagcgc agaacaaatg ctgaagggcc acaaagaagt attgattttc

661 ggtggtcagg aagtggaaac gaaacgcttt gataaagtca gcaaccgaat gcgtcttcag

721 gggatgaaaa tggtttcagc ctcttccatc tctgatccga tcattcagct gatcgcctct

781 ttggcgctgg cgtttgttct gtatgcggcg agcttcccaa gtgtcatgga tagcctgact

841 gccggtacga ttaccgttgt tttctcttca atgattgcac tgatgcgtcc gctgaaatcg

901 ctgaccaacg ttaacgccca gttccagcgc ggtatggcgg cttgtcagac gctgtttacc

961 attctggaca gtgagcagga gaaagatgaa ggtaagcgcg tgatcgagcg tgcgactggc

1021 gacgtggaat tccgcaatgt cacctttact tatccgggac gtgacgtacctgcattgcgt

1081 aacatcaacc tgaaaattcc ggcagggaag acggttgctc tggttggacgctctggttcg

1141 ggtaaatcaa ccatcgccag cctgatcacg cgtttttacg atattgatgaaggcgaaatc

1201 ctgatggatg gtcacgatct gcgcgagtat accctggcgt cgttacgtaaccaggttgct

1261 ctggtgtcgc agaatgtcca tctgtttaac gatacggttg ctaacaacattgcttacgca

1321 cggactgaac agtacagccg tgagcaaatt gaagaagcgg cgcgtatggcctacgccatg

1381 gacttcatca ataagatgga taacggtctc gatacagtga ttggtgaaaacggcgtgctg

1441 ctctctggcg gtcagcgtca gcgtattgct atcgctcgag ccttgttgcgtgatagcccg

1501 attctgattc tggacgaagc tacctcggct ctggataccg aatccgaacgtgcgattcag

1561 gcggcactgg atgagttgca gaaaaaccgt acctctctgg tgattgcccaccgcttgtct

1621 accattgaaa aggcagacga aatcgtggtc gtcgaggatg gtgtcattgtggaacgcggt

1681 acgcataacg atttgcttga gcaccgcggc gtttacgcgc aacttcacaaaatgcagttt

1741 ggccaatga

Claims (5)

1. The low endotoxin escherichia coli subjected to high-density fermentation is characterized by being classified and named as Escherichia coli CleanTAK M and preserved in China Center for Type Culture Collection (CCTCC) M20221906, wherein the preservation number is 2022, and the preservation time is 12 months and 09 days;

on the basis of a CleanTAK6 alpha engineering strain, the constructed target gene recombinant pTarget plasmid and the fused homology arm fragment are electrically transferred into cells of the CleanTAK6 alpha engineering strain containing pCas plasmid, and the CleanTAK7 engineering strain is constructed, wherein the knocked-out gene combination is as follows: ΔeptaΔkdsdΔpagpΔlpxlΔlpxpΔlpxmΔgutq;

the CleanTAK6 alpha is an engineering strain for knocking out eptA, kdsD, pagP, lpxL, lpxP and lpxM genes;

the construction of the CleanTAK 7m engineering strain comprises the following steps:

1) Construction of pTarget-sgRNA-msbA recombinant plasmid:

the 5' -NGG sequence is closely searched at the position to be mutated of the msbA gene, 31bp upstream of the NGG sequence is used as a targeting sequence of sgRNA, and the following primers which contain 20bp and have 20-25bp homologous fragments with pTarget plasmid are designed:

msbA-sg-IF：

5’GATCTGTTTTACCAAAGCCATCATCAAGAAGGTTTTAGAGCTAGAAATA GCAAGTT 3’

msbA-sg-IR：5’CGATCAACGGCACTGTTGCAAATAG 3’

msbA-sg-VF：5’CTATTTGCAACAGTGCCGTTGATCG 3’

msbA-sg-VR：

5’TTCTTGATGATGGCTTTGGTAAAACAGATCACTAGTATTATACCTAGGAC TGAGC 3’

respectively carrying out PCR amplification by taking pTarget plasmid DNA as a template to construct an insert fragment and a carrier fragment of the recombinant plasmid;

2) Amplification and fusion of msbA gene point mutation site-containing fragments

Designing two pairs of fusion PCR primers to amplify exogenous fragments required by msbA gene point mutation respectively, wherein the sequences of the primers are as follows:

msbA-S1-F:5’AAAAGTGGCACTCGCATCGG 3’

msbA-S1-R:

5’CGATCTGTTTTACCAAAGCCATCATCAAGAAGTGACTTAAG

GAGCGATAACATGAAGGT 3’

msbA-S2-F:

5’ACCTTCATGTTATCGCTCCTTAAGTCACTTCTTGATGATGG

CTTTGGTAAAACAGATCG 3’

msbA-S2-R:5’AAAACGCTTCGATACAACGC 3’

wherein the msbA-S1-R and msbA-S2-F primers comprise a mutated base, i.e., the 35 th base is replaced by G/C to a/T;

fusion of S1 and S2 fragments: adding 1 mu L of each of the amplified S1 and S2 fragments as a template of overlap extension PCR, and amplifying the fragments by using primers msbA-S1-F and msbA-S2-R under an amplification system to obtain fusion fragments;

3) Construction and identification of msbA gene point mutation engineering strain

Electrotransferring the constructed pTarget-sgRNA-msbA recombinant plasmid and the fused exogenous fragment into an escherichia coli CleanTAK7 (pCas) strain, coating the strain onto a plate with spectinomycin and kanamycin double resistance, and placing the plate in a 30 ℃ incubator for overnight culture; single colony is streaked on a new double-antibody plate for culture, and then genome is extracted as a template for PCR amplification verification, wherein the PCR amplification reaction is carried out in a 20 mu L system, and the reaction system is as follows: template DNA 2. Mu.L, 2 XHi-Fi DNase premix 10. Mu.L, upstream primer 1. Mu.L, downstream primer 1. Mu.L, ddH ₂ O6. Mu.L; the primer sequences used were as follows:

U-msbA-F:5’TAACGGGTAGAATATGCGGC 3’

U-msbA-R:5’CTGGCATTCCCATCATGTGA 3’

sequencing and analyzing the PCR product with the band size meeting the expectations, comparing the sequencing result with the TAK strain genome, confirming the successful point mutation of the msbA gene, and naming the engineering strain as CleanTAK 7m;

4) Elimination of plasmids in CleanTAK 7m engineering strains

The clearTAK 7m engineering strain containing pTarget and pCas double plasmid is transferred into 5mL LB liquid medium containing Kan (50 mug/mL) resistance, then IPTG with a final concentration of 1mmol/L is added to induce the sgRNA expression on pCas plasmid, and the mixture is cultured overnight in a shaker at 30 ℃ and 180r/min, thus obtaining the clearTAK 7m engineering strain without plasmid.

2. Use of the low endotoxin e.coli as claimed in claim 1 for expression of protein/polypeptide substances.

3. Use of the low endotoxin e.coli as claimed in claim 1 for the preparation of an enzyme product.

4. Use of the low endotoxin e.coli as claimed in claim 1 for the preparation of plasmids.

5. Use of the low endotoxin e.coli of claim 1 for the preparation of secondary metabolites for the chassis of synthetic biology.